Instruction Manual. plenti6/tr Vector. Catalog no. V Version C 31 October

Transcription

1 Instruction Manual plenti6/tr Vector Catalog no. V Version C 31 October

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3 Table of Contents Table of Contents...iii Kit Contents and Storage.... v Accessory Products...vi Introduction....1 Overview... 1 How Tetracycline Regulation Works... 7 Biosafety Features Methods General Considerations to Use plenti6/tr Guidelines for Transfection Generating a Stable Cell Line Via Plasmid Transfection Guidelines to Produce a Lentiviral Stock Appendix Map and Features of plenti6/tr Blasticidin Technical Service Purchaser Notification Product Qualification References iii

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5 Kit Contents and Storage Shipping/ Storage The plenti6/tr Vector Kit is shipped in two boxes as described below. Upon receipt, store as recommended below. Box Reagent Shipping Storage 1 plenti6/tr Vector Room temperature -20 C 2 Blasticidin Room temperature -20 C Contents The plenti6/tr Vector Kit includes the following reagents. Store at -20 C. Reagent Composition Amount plenti6/tr Vector Lyophilized in TE Buffer, ph µg Blasticidin Powder 50 mg v

6 Accessory Products Accessory Products Blasticidin and other products suitable for use with the plenti6/tr Vector Kit are available separately from Invitrogen. Ordering information is provided below. For more information, refer to our Web site ( or call Technical Service (see page 26). Note: Some reagents are available in other sizes. Item Amount Catalog no. Blasticidin 50 mg R ViraPower Packaging Mix 3 x 195 µg K FT Cell Line 3 x 10 6 cells, frozen R Lipofectamine 2000 Reagent 0.75 ml ml Opti-MEM I Reduced Serum Medium One Shot Stbl3 Chemically Competent E. coli 100 ml 500 ml x 50 µl C Invitrogen Expression Systems The plenti6/tr vector is intended for use in generating stable cell lines expressing the Tet repressor. These TetRexpressing cell lines may be used as hosts for expression constructs that facilitate tetracycline-regulated expression of a gene or shrna of interest from Invitrogen s ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System, respectively. Ordering information for these two systems is provided below. Item Amount Catalog no. ViraPower T-REx Lentiviral 20 constructions K Expression System BLOCK-iT Inducible H1 Lentiviral RNAi System 20 constructions K vi

7 Introduction Overview Introduction plenti6/tr is an 8.4 kb lentiviral-based vector that facilitates high-level expression of the tetracycline (Tet) repressor under the control of the human cytomegalovirus (CMV) immediate early promoter. The vector also contains elements that allow packaging of the construct into virions and the Blasticidin resistance marker for selection of stable cell lines. plenti6/tr is designed for use with the ViraPower T-REx Lentiviral Expression System (Catalog nos. K and K ) and the BLOCK-iT Inducible H1 Lentiviral RNAi System (Catalog no. K ) available from Invitrogen. Lenti6/TR lentivirus may be transduced into dividing or nondividing mammalian cells to facilitate high-level stable or transient expression of the Tet repressor. Tetracyclineregulated expression of a gene or short hairpin RNA (shrna) of interest may then be tested by transducing the appropriate inducible lentiviral expression construct into host cells expressing the Tet repressor. For more information about the ViraPower Lentiviral Technology, ViraPower T-REx Lentiviral Expression System, and the BLOCK-iT Inducible H1 Lentiviral RNAi System, see below and pages 2-3. For a brief description about how tetracycline regulation works in these Systems, see page 7. ViraPower Lentiviral Technology The ViraPower Lentiviral Technology facilitates highly efficient, in vitro or in vivo delivery of a target gene or RNA to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat system developed by Cell Genesys (Dull et al., 1998), the ViraPower Lentiviral Technology possesses features which enhance its biosafety while allowing high-level expression in a wider range of cell types than traditional retroviral systems. For more information about the biosafety features of the System, see page 8. continued on next page 1

8 Overview, continued ViraPower T-REx Lentiviral Expression System The ViraPower T-REx Lentiviral Expression System combines Invitrogen s ViraPower Lentiviral and T-REx technologies to facilitate highly efficient, in vitro or in vivo tetracycline-regulated expression of a target gene of interest in dividing or non-dividing mammalian cells using a replication-incompetent lentivirus. The System includes: The plenti6/tr vector containing the TetR gene for constitutive, high-level expression of the Tet repressor under the control of a CMV promoter (see page 5 for more information). The plenti4/to/v5-dest Gateway destination vector into which the gene of interest is cloned. The vector contains elements to allow packaging of the construct into virions and tetracycline-regulated expression of the gene of interest. The ViraPower Packaging Mix and the 293FT Cell Line to facilitate production of replication-incompetent lentiviral stocks from any plenti-based construct. Tetracycline to induce expression of the gene of interest. For more information about the Gateway Technology, see page 4. For more information about the ViraPower T-REx Lentiviral Expression System, refer to the ViraPower T-REx Lentiviral Expression System manual. This manual is available for downloading from our Web site ( or by contacting Technical Service (see page 26). continued on next page 2

9 Overview, continued BLOCK-iT Inducible H1 Lentiviral RNAi System The BLOCK-iT Inducible H1 Lentiviral RNAi System combines Invitrogen s BLOCK-iT RNAi and ViraPower T-REx Lentiviral technologies to facilitate lentiviral-based, tetracycline-regulated expression of a short hairpin RNA (shrna) of interest in dividing or non-dividing mammalian cells for RNA interference (RNAi) analysis. The System includes: The BLOCK-iT Inducible H1 RNAi Entry Vector Kit which includes the pentr /H1/TO vector for production of a Gateway entry clone containing elements required for tetracycline-regulated expression of a double-stranded oligonucleotide encoding an shrna of interest in mammalian cells. This H1/TO RNAi cassette (i.e. human H1/TO promoter + doublestranded oligonucleotide + Polymerase III terminator) can be transferred into the plenti4/block-it -DEST vector in an LR recombination reaction. The promoterless plenti4/block-it -DEST Gateway destination vector into which the H1/TO RNAi cassette is transferred. The vector contains elements to allow packaging of the construct into virions and the Zeocin resistance gene for selection of stable cell lines. The plenti6/tr vector containing the TetR gene for constitutive, high-level expression of the Tet repressor under the control of a CMV promoter (see page 5 for more information). The ViraPower Packaging Mix and the 293FT Cell Line to facilitate production of replication-incompetent lentiviral stocks from any plenti-based construct. Tetracycline to induce expression of the gene of interest. For more information about the Gateway Technology, see the next page. For more information about the BLOCK-iT Inducible H1 Lentiviral RNAi System, refer to the BLOCK-iT Inducible H1 Lentiviral RNAi System manual. This manual is available for downloading from our Web site ( or by contacting Technical Service (see page 26). continued on next page 3

10 Overview, continued Gateway Technology Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your DNA sequence of interest into multiple vector systems. In the ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System, the gene or double-stranded oligonucleotide of interest, respectively, is cloned into an appropriate Gateway -adapted entry vector to generate an entry clone. The resulting entry clone is then used in an LR recombination reaction with the appropriate Gateway destination vector (e.g. plenti4/to/v5-dest or plenti4/block-it -DEST) to generate an expression clone containing the gene or H1/TO RNAi cassette of interest, respectively. For detailed information about the Gateway Technology, refer to the Gateway Technology manual which is available from our Web site ( or by contacting Technical Service (see page 26). 4

11 Overview, continued Features of the plenti6/tr Vector The plenti6/tr vector contains the following elements: Rous Sarcoma Virus (RSV) enhancer/promoter for Tatindependent production of viral mrna in the producer cell line (Dull et al., 1998) Modified HIV-1 5 and 3 Long Terminal Repeats (LTR) for viral packaging and reverse transcription of the viral mrna (Dull et al., 1998; Luciw, 1996) Note: The U3 region of the 3 LTR is deleted to enhance the biosafety of the vector. For more information about the biosafety features of the vector, see page 8. HIV-1 psi (Ψ) packaging sequence for viral packaging (Luciw, 1996) HIV Rev response element (RRE) for Rev-dependent nuclear export of unspliced viral mrna (Kjems et al., 1991; Malim et al., 1989) Human CMV promoter for high-level, constitutive expression of the TetR gene Rabbit β-globin intron II sequence for enhanced expression of the TetR gene in cultured cells (van Ooyen et al., 1979) TetR gene encoding the Tet repressor to repress transcription of your gene in the absence of tetracycline (Postle et al., 1984; Yao et al., 1998) Blasticidin resistance gene (Izumi et al., 1991; Kimura et al., 1994; Takeuchi et al., 1958; Yamaguchi et al., 1965) for selection in E. coli and mammalian cells Ampicillin resistance gene for selection in E. coli puc origin for high-copy replication of the plasmid in E. coli For a map of plenti6/tr, see the Appendix, page 21. continued on next page 5

12 Overview, continued TetR Gene in plenti6/tr The TetR gene in plenti6/tr was originally isolated from the Tn10 transposon which confers resistance to tetracycline in E. coli and other enteric bacteria (Postle et al., 1984). The TetR gene from Tn10 encodes a class B Tet repressor and is often referred to as TetR(B) in the literature (Hillen and Berens, 1994). The TetR gene encodes a repressor protein of 207 amino acids with a calculated molecular weight of 23 kda. For more information about the Tet repressor and its interaction with the Tet operator, refer to Hillen and Berens, Producing Lentivirus To use plenti6/tr to generate a TetR-expressing cell line, you will need to produce a lentiviral stock. To produce a lentiviral stock, you will cotransfect the ViraPower Packaging Mix and the plenti6/tr vector into 293FT producer cells to produce replication-incompetent lentivirus. The Lenti6/TR lentivirus can then be transduced into the mammalian cell line of interest and Blasticidin selection can be performed to select for stable cell lines. For more information about the ViraPower Packaging Mix and the 293FT Cell Line, see page 19. How Lentivirus Works Once the lentivirus enters the target cell, the viral RNA is reverse-transcribed, actively imported into the nucleus (Lewis and Emerman, 1994; Naldini, 1999), and stably integrated into the host genome (Buchschacher and Wong- Staal, 2000; Luciw, 1996). Once the lentiviral construct has integrated into the genome, the TetR gene is expressed. VSV Envelope Glycoprotein Most retroviral vectors are limited in their usefulness as delivery vehicles by their restricted tropism and generally low titers. In the ViraPower and BLOCK-iT Lentiviral Systems, this limitation has been overcome by use of the G Glycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as a pseudotyping envelope, thus allowing production of a high titer lentivirus with a significantly broadened host cell range (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994). 6

13 How Tetracycline Regulation Works Tetracycline Regulation The ViraPower T-REx Lentiviral Expression System and the BLOCK-iT Inducible H1 Lentiviral RNAi System use regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens, 1994; Hillen et al., 1983) to allow tetracycline-regulated expression of a gene or shrna of interest, respectively from an appropriate inducible lentiviral construct. The mechanism of tetracycline regulation in the system is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest (Yao et al., 1998). In each system, expression of the molecule of interest is repressed in the absence of tetracycline and induced in its presence (Yao et al., 1998). In each system, expression of the gene or shrna of interest from the plenti4/to/v5-dest or plenti4/block-it - DEST lentiviral construct, respectively is controlled by a hybrid promoter containing 2 copies of the tet operator 2 (TetO2) sequence. Each 19-nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor. Mechanism of Repression/ Derepression In the absence of tetracycline, the Tet repressor (expressed from plenti6/tr) forms a homodimer that binds with extremely high affinity to each TetO2 sequence (Hillen and Berens, 1994) in the promoter of the plenti4/to/v5-dest or plenti4/block-it -DEST expression construct. The 2 TetO2 sites in the promoter of each construct serve as binding sites for 4 molecules (or 2 homodimers) of the Tet repressor. Binding of the Tet repressor homodimers to the TetO2 sequences represses transcription of your gene or shrna of interest. Upon addition, tetracycline binds with high affinity to each Tet repressor homodimer in a 1:1 stoichiometry and causes a conformational change in the repressor that renders it unable to bind the Tet operator. The Tet repressor:tetracycline complex then dissociates from the Tet operator and allows induction of transcription of the gene or shrna of interest. Note: The affinity of the Tet repressor for the tet operator is KB = 2 x M -1 (as measured under physiological conditions), where KB is the binding constant. The association constant, KA, of tetracycline for the Tet repressor is 3 x 10 9 M -1 (Hillen and Berens, 1994). 7

14 Biosafety Features Introduction The lentiviral and packaging vectors supplied in the ViraPower T-REx Lentiviral Expression System and the BLOCK-iT Inducible H1 Lentiviral RNAi System are thirdgeneration vectors based on lentiviral vectors developed by Dull et al., This third-generation HIV-1-based lentiviral system includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild-type, human HIV-1 virus. These safety features are discussed below. Biosafety Features of Invitrogen s Lentiviral Systems Invitrogen s ViraPower T-REx and BLOCK-iT Inducible H1 RNAi Lentiviral Systems include the following key safety features: All plenti-based vectors contain a deletion in the 3 LTR ( U3) that does not affect generation of the viral genome in the producer cell line but results in self-inactivation of the lentivirus after transduction of the target cell (Yee et al., 1987; Yu et al., 1986; Zufferey et al., 1998). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome. The number of genes from HIV-1 that are used in the system has been reduced to three (i.e. gag, pol, and rev). The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994). Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids (i.e. three packaging plasmids and the plenti-based plasmid). All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replicationcompetent virus (Dull et al., 1998). continued on next page 8

15 Biosafety Features, continued Biosafety Features of Invitrogen s Lentiviral Systems, continued Although the three packaging plasmids allow expression in trans of proteins required to produce viral progeny (e.g. gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the Ψ packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced. The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. No other viral species are produced. Expression of the gag and pol genes from plp1 has been rendered Rev-dependent by virtue of the HIV-1 RRE in the gag/pol mrna transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Dull et al., 1998). A constitutive promoter (RSV promoter) has been placed upstream of the 5 LTR in each plenti-based vector to offset the requirement for Tat in the efficient production of viral RNA (Dull et al., 1998). continued on next page 9

16 Biosafety Features, continued Biosafety Level 2 Despite the inclusion of the safety features discussed in this section, the lentivirus produced with the ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral stocks generated using either System as Biosafety Level 2 (BL-2) organisms and strictly follow all published BL-2 guidelines with proper waste decontamination. Furthermore, exercise extra caution when creating lentivirus carrying potentially harmful or toxic genes (e.g. activated oncogenes) or shrna molecules targeting human tumor suppressor genes. For more information about the BL-2 guidelines and lentivirus handling, refer to the document, Biosafety in Microbiological and Biomedical Laboratories, 4 th Edition, published by the Centers for Disease Control (CDC). This document may be downloaded from the Web at Important Handle all lentiviruses in compliance with established institutional guidelines. Since safety requirements for use and handling of lentiviruses may vary at individual institutions, we recommend consulting the health and safety guidelines and/or officers at your institution prior to use of the ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System. 10

17 Methods General Considerations to Use plenti6/tr Introduction This section provides general guidelines and discusses the options available to use the plenti6/tr vector to express Tet repressor in a mammalian cell line of interest. Options to Use plenti6/tr Several options are available to use plenti6/tr to obtain transient or stable expression of the Tet repressor. Choose the option that best fits your needs. Option Procedure Benefit 1 Produce a lentiviral stock of the plenti6/tr construct, then cotransduce the Lenti6/TR and the inducible Lenti-DEST-based lentiviral construct into the mammalian cell line of choice. 2 Produce a lentiviral stock of the plenti6/tr construct, then transduce the mammalian cell line of choice with the Lenti6/TR lentivirus and use Blasticidin selection to generate a stable cell line. Use this TetR-expressing cell line as the host for the inducible Lenti-DEST-based lentiviral construct. 3 Transfect the plenti6/tr plasmid into mammalian cells and use Blasticidin selection to generate a stable cell line. Use this TetRexpressing cell line as the host for the inducible Lenti-DEST-based lentiviral construct. Perform tetracycline-regulated expression experiments with a single transduction. Note: Suitable for dividing and non-dividing mammalian cell lines. Perform tetracycline-regulated experiments with multiple constructs using a cell line that consistently expresses the same amount of Tet repressor. Perform tetracycline-regulated experiments in a dividing mammalian cell line only. continued on next page 11

18 General Considerations to Use plenti6/tr, continued Resuspending the Plasmid The plenti6/tr plasmid is supplied lyophilized in TE Buffer, ph 8.0. To use, resuspend the DNA in 20 µl of sterile water to prepare a 1 µg/µl stock solution. Store stock solution at -20 C. Note: If you wish to propagate the plenti6/tr plasmid, see below. E. coli Host For optimal results, we recommend using Stbl3 E. coli for transformation, as this strain is particularly well-suited for cloning unstable DNA such as lentiviral DNA containing direct repeats. Other E. coli strains such as TOP10 or DH5α are suitable for transformation but may give rise to some transformants containing unwanted recombinants (i.e. plasmids where recombination has occurred between the 5 and 3 LTRs). To reduce the frequency of this occurrence, select for TOP10 and DH5α transformants on selective plates containing ampicillin and Blasticidin. Propagating the plenti6/tr Plasmid The plenti6/tr vector contains the ampicillin and Blasticidin resistance genes to facilitate selection of the plasmid in E. coli. To propagate and maintain the plasmid: 1. Use the 1 µg/µl plenti6/tr stock solution (see above) to transform a reca, enda E. coli strain such as Stbl3, TOP10, or DH5α. Use 10 ng of plasmid for transformation. 2. Depending on the E. coli strain transformed, select transformants on plates containing the following: If you are transforming Stbl3 TOP10 or DH5α Then use LB agar plates containing 100 µg/ml ampicillin LB agar plates containing 100 µg/ml ampicillin and 50 µg/ml Blasticidin 3. Prepare glycerol stocks of a transformant containing plasmid for long-term storage. Propagate the plasmid in LB medium containing 100 µg/ml ampicillin. 12

19 Guidelines for Transfection Introduction To generate a stable cell line expressing Tet repressor from plenti6/tr, you may either: Transfect the plenti6/tr plasmid directly into a dividing mammalian cell line and use Blasticidin to select for a stable cell line OR Cotransfect the plenti6/tr plasmid and the ViraPower Packaging Mix into the 293FT producer cell line to produce a lentiviral stock, then transduce the resulting lentiviral stock into any mammalian cell line of choice and use Blasticidin selection to generate a stable cell line General guidelines are provided in this section to perform transfection. We suggest reading through this section before beginning. For instructions to generate a stable cell line via plasmid transfection, see page 16. For guidelines to produce a lentiviral stock, see page 18. Plasmid Preparation Once resuspended, the plenti6/tr supplied in the kit is ready-to-use for transfection. If you are propagating the plenti6/tr plasmid, note that you must use purified plasmid DNA for transfection. Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination with phenol or sodium chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the PureLink HQ Mini Plasmid Purification Kit (Catalog no. K ) or the S.N.A.P. MidiPrep Kit (Catalog no. K ) available from Invitrogen or CsCl gradient centrifugation. continued on next page 13

20 Guidelines for Transfection, continued Methods of Transfection Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989), and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). For established cell lines (e.g. 293, COS, A549), consult original references or the supplier of your cell line for the optimal method of transfection. Choose the method and reagent that provides the highest efficiency transfection in your mammalian cell line. We recommend using Invitrogen s Lipofectamine 2000 Reagent for transfection (see below). Important: If you are producing a lentiviral stock, you must use Lipofectamine 2000 for transfection. Lipofectamine 2000 For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using the cationic lipidbased Lipofectamine 2000 Reagent (Catalog no ) available from Invitrogen (Ciccarone et al., 1999). Using Lipofectamine 2000 to transfect plasmid DNA into eukaryotic cells offers the following advantages: Provides the highest transfection efficiency in many mammalian cell types. DNA-Lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence of serum. Removal of complexes, medium change, or medium addition following transfection is not required, although complexes can be removed after 4-6 hours without loss of activity. For more information on Lipofectamine 2000 Reagent, refer to our Web site ( or call Technical Service (see page 26). continued on next page 14

21 Guidelines for Transfection, continued Determining Blasticidin Sensitivity for Your Cell Line The plenti6/tr vector contains the Blasticidin resistance gene to allow generation of stable cell lines using Blasticidin. Before you can generate a stable cell line, you must determine the minimum concentration of Blasticidin required to kill your untransfected mammalian cell line (i.e. perform a kill curve experiment). Typically, concentrations ranging from 2-10 µg/ml Blasticidin are sufficient to kill most untransfected mammalian cell lines. We recommend that you test a range of concentrations to ensure that you determine the minimum concentration necessary for your cell line. For instructions to prepare and handle Blasticidin, see the Appendix, page Plate cells at approximately 25% confluence. Prepare a set of 6-7 plates. Allow cells to adhere overnight. 2. The next day, substitute culture medium with medium containing varying concentrations of Blasticidin. 3. Replenish the selective media every 3-4 days and observe the percentage of surviving cells. 4. Determine the appropriate concentration of Blasticidin that kills the cells within days after addition of antibiotic. 15

22 Generating a Stable Cell Line Via Plasmid Transfection Introduction This section provides general guidelines to transfect the plenti6/tr plasmid into the mammalian cell line of interest and to use Blasticidin selection to generate a stable cell line. Important Because tetracycline-regulated expression in the ViraPower T-REx Lentiviral Expression System and the BLOCK-iT Inducible H1 Lentiviral RNAi System is based on a repression/derepression mechanism, the amount of Tet repressor expressed in the host cell line from the Lenti6/TR lentiviral construct will determine the level of transcriptional repression of the TetO2 sequences in the inducible lentiviral construct. When generating stable TetR-expressing cell lines, note that Tet repressor levels need to be sufficiently high to suitably repress basal level transcription of the gene or shrna of interest, as appropriate. Materials Needed To transfect the plenti6/tr plasmid into mammalian cells, you will need to have the following materials on hand: Mammalian cell line of interest (make sure that cells are healthy and > 90% viable before beginning) plenti6/tr plasmid (1 µg/µl stock solution) Transfection reagent of choice (e.g. Lipofectamine 2000) 10 mg/ml Blasticidin Appropriate growth medium for your cells Appropriate tissue culture dishes and supplies continued on next page 16

23 Generating a Stable Cell Line Via Plasmid Transfection, continued Guidelines for Transfection and Selection Follow these guidelines to transfect the plenti6/tr plasmid into mammalian cells and to select for stable cell lines using Blasticidin. Important: Do not use these guidelines if you are preparing a lentiviral stock. See the next section for guidelines to produce a lentiviral stock. 1. One day before transfection, plate cells at a density recommended by the manufacturer of the transfection reagent you are using. 2. On the day of transfection (Day 1), transfect plenti6/tr plasmid into cells following the recommendations of the manufacturer of your transfection reagent. 3. Six hours after transfection, remove the medium and replace with fresh growth medium. Incubate the cells overnight at 37 C. 4. The following day (Day 2), remove the medium and replace with fresh complete medium containing the appropriate concentration of Blasticidin. 5. Replace medium with fresh medium containing Blasticidin every 3-4 days until Blasticidin-resistant colonies can be identified (generally days after selection). 6. Pick at least 10 Blasticidin-resistant colonies. Screen to determine the amount of Tet repressor expressed. Select clones that express the highest levels of Tet repressor to use as hosts for your inducible expression construct. Detecting TetR Protein To detect Tet repressor expression, we recommend performing Western blot analysis using an Anti-Tet repressor antibody (MoBiTec, Göttingen, Germany, Catalog no. TET01). Maintaining TetR- Expressing Cell Lines Once you have generated your stable TetR-expressing cell line and have verified that the cells express suitable levels of Tet repressor, we recommend the following: Maintain the cell line in medium containing Blasticidin Freeze and store vials of early passage cells 17

24 Guidelines to Produce a Lentiviral Stock Introduction This section provides general guidelines to produce a lentiviral stock from plenti6/tr. Once produced, you may: Transduce the Lenti6/TR lentivirus into the mammalian cells of choice and use Blasticidin to select for a stable cell line. Use the resulting TetR-expressing cell line as the host for your inducible lentiviral construct. Co-transduce the Lenti6/TR lentiviral construct and the inducible lentiviral construct into the mammalian cells of choice and perform transient or stable expression, as appropriate. For detailed instructions and protocols to produce a lentiviral stock and transduce mammalian cells, refer to the ViraPower T-REx Lentiviral Expression System manual or the BLOCK-iT Inducible H1 Lentiviral RNAi System manual. Both manuals are available for downloading from our Web site or by contacting Technical Service. Experimental Outline To generate a stable TetR-expressing cell line using lentivirus, you will need to do the following: 1. Cotransfect the plenti6/tr construct and the ViraPower Packaging Mix into the 293FT Cell Line to produce a lentiviral stock. 2. Titer the lentiviral stock. 3. Transduce the Lenti6/TR lentivirus into the mammalian cells of choice at an appropriate multiplicity of infection (MOI). 4. Use Blasticidin to select for stably transduced cells. 5. Pick Blasticidin-resistant clones and screen for clones that express the highest levels of Tet repressor (see Important Note on page 16). For more information about the ViraPower Packaging Mix and the 293FT Cell Line, see the next page. continued on next page 18

25 Guidelines to Produce a Lentiviral Stock, continued ViraPower Packaging Mix The ViraPower Packaging Mix (Catalog no. K ) contains an optimized mixture of three packaging plasmids, plp1, plp2, and plp/vsvg. These plasmids supply the helper functions as well as structural and replication proteins in trans required to facilitate viral packaging of the plenti6/tr construct following cotransfection into 293FT producer cells. For more information about the ViraPower Packaging Mix and each packaging plasmid, refer to the ViraPower T-REx Lentiviral Expression System or BLOCKiT Inducible H1 Lentiviral RNAi System manual. 293FT Cell Line The human 293FT Cell Line (Catalog no. R700-07) facilitates optimal production of lentivirus (Naldini et al., 1996) and is derived from the 293F cell line. The 293FT Cell Line stably expresses the SV40 large T antigen under the control of the human CMV promoter and must be maintained in medium containing Geneticin. For more information about the 293FT Cell Line including instructions to culture and maintain the cells, refer to the 293FT Cell Line manual, which is available for downloading from our Web site ( or by calling Technical Service (see page 26). continued on next page 19

26 Guidelines to Produce a Lentiviral Stock, continued Materials Needed You will need the following reagents to produce a lentiviral stock from plenti6/tr: plenti6/tr plasmid (1 µg/µl stock solution) ViraPower Packaging Mix (Catalog no. K ) 293FT cells cultured in complete growth medium (see the 293FT Cell Line manual for details; Catalog no. R700-07) Lipofectamine 2000 transfection reagent (Catalog no ) Opti-MEM I Reduced Serum Medium (recommended for optimal DNA-Lipofectamine 2000 complex formation; Catalog no ) Appropriate growth medium Appropriate tissue culture dishes and supplies For details about the appropriate growth medium to use for lentiviral production, refer to the ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System manual. Producing Lentiviral Stocks For detailed guidelines and instructions to produce a Lenti6/TR lentiviral stock, titer the lentivirus, and transduce mammalian cells, refer to the ViraPower T-REx Lentiviral Expression System or the BLOCK-iT Inducible H1 Lentiviral RNAi System manual. 20

27 Appendix Map and Features of plenti6/tr Map of plenti6/tr The map below shows the elements of plenti6/tr. The complete sequence for plenti6/tr is available on our Web site ( or by contacting Technical Service (see page 26). b-globin IVS tetr Stop RRE P CMV P SV40 EM7 y Comments for plenti6/tr 8355 nucleotides /5 LTR RSV/5 LTR hybrid promoter: bases RSV promoter: bases HIV-1 5 LTR: bases splice donor: base 520 HIV-1 psi (y) packaging signal: bases HIV-1 Rev response element (RRE): bases splice acceptor: base splice acceptor: base 1684 CMV promoter: bases Rabbit b-globin intron II (IVS): bases tetr gene: bases SV40 early promoter and origin: bases EM7 promoter: bases Blasticidin resistance gene: bases DU3/3 LTR: bases DU3: bases LTR: bases SV40 polyadenylation signal: bases bla promoter: bases Ampicillin (bla) resistance gene: bases puc origin: bases PRSV puc ori plenti6/tr 8355 bp Ampicillin SV40 pa Blasticidin DU3/3 LTR 21

28 Map and Features of plenti6/tr, continued Features of the Vector The plenti6/tr (8355 bp) vector contains the following elements. All features have been functionally tested. Feature Rous Sarcoma Virus (RSV) enhancer/promoter HIV-1 truncated 5 LTR Benefit Allows Tat-independent production of viral mrna (Dull et al., 1998). Allows viral packaging and reverse transcription of the viral mrna (Luciw, 1996). 5 splice donor and 3 acceptors Enhances the biosafety of the vector by facilitating removal of the Ψ packaging sequence and RRE such that expression of the gene of interest in the transduced host cell is no longer Rev-dependent (Dull et al., 1998). HIV-1 psi (ψ) packaging signal Allows viral packaging (Luciw, 1996). HIV-1 Rev response element (RRE) CMV promoter Rabbit β-globin intron II (IVS) TetR gene SV40 early promoter and origin EM7 promoter Allows Rev-dependent nuclear export of unspliced viral mrna (Kjems et al., 1991; Malim et al., 1989). Allows high-level, constitutive expression of the Tet repressor in mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987). Enhances expression of the TetR gene in mammalian cells (van Ooyen et al., 1979). Encodes the Tet repressor that binds to tet operator sequences to repress transcription of the gene of interest in the absence of tetracycline (Postle et al., 1984; Yao et al., 1998). Allows high-level expression of the selection marker and episomal replication in cells expressing the SV40 large T antigen. Synthetic prokaryotic promoter for expression of the selection marker in E. coli. continued on next page 22

29 Map and Features of plenti6/tr, continued Features of the Vector, continued Feature Blasticidin (bsd) resistance gene U3/HIV-1 truncated 3 LTR SV40 polyadenylation signal bla promoter Ampicillin resistance gene (β-lactamase) puc origin Benefit Allows selection of stably transduced mammalian cell lines (Kimura et al., 1994). Allows viral packaging but self-inactivates the 5 LTR for biosafety purposes (Dull et al., 1998). The element also contains a polyadenylation signal for transcription termination and polyadenylation of mrna in transduced cells. Allows transcription termination and polyadenylation of mrna. Allows expression of the ampicillin resistance gene. Allows selection of the plasmid in E. coli. Allows high-copy replication and maintenance in E. coli. 23

30 Blasticidin Blasticidin Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two Blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert Blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991). Molecular Weight, Formula, and Structure The formula for Blasticidin S is C17H26N8O5-HCl, and the molecular weight is The diagram below shows the structure of Blasticidin. NH2 N CH3 HOOC O N O -HCl H2N N NH NH NH2 O Handling Blasticidin Always wear gloves, mask, goggles, and protective clothing (e.g. a laboratory coat) when handling Blasticidin. Weigh out Blasticidin and prepare solutions in a hood. continued on next page 24

31 Blasticidin, continued Preparing and Storing Stock Solutions Blasticidin may be obtained separately from Invitrogen (Catalog no. R210-01) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 to 10 mg/ml. Dissolve Blasticidin in sterile water and filter-sterilize the solution. Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at -20 C for longterm storage or store at +4 C for short-term storage. Aqueous stock solutions are stable for 1-2 weeks at +4 C and 6-8 weeks at -20 C. ph of the aqueous solution should be 7.0 to prevent inactivation of Blasticidin. Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer). Upon thawing, use what you need and store the thawed stock solution at +4 C for up to 2 weeks. Medium containing Blasticidin may be stored at +4 C for up to 2 weeks. 25

32 Technical Service World Wide Web Visit the Invitrogen Web site at for: Technical resources, including manuals, vector maps and sequences, application notes, MSDSs, FAQs, formulations, citations, handbooks, etc. Complete technical service contact information Access to the Invitrogen Online Catalog Additional product information and special offers Contact Us Corporate Headquarters: Invitrogen Corporation 1600 Faraday Avenue Carlsbad, CA USA Tel: Tel (Toll Free): Fax: tech_service@invitrogen.com For more information or technical assistance, call, write, fax, or . Additional international offices are listed on our Web page ( Japanese Headquarters: Invitrogen Japan LOOP-X Bldg. 6F , Kaigan Minato-ku, Tokyo Tel: Fax: jpinfo@invitrogen.com European Headquarters: Invitrogen Ltd Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF, UK Tel: +44 (0) Tech Fax: +44 (0) eurotech@invitrogen.com Material Data Safety Sheets (MSDSs) MSDSs are available on our Web site at On the home page, click on Technical Resources and follow instructions on the page to download the MSDS for your product. continued on next page 26

33 Technical Service, continued Limited Warranty Invitrogen is committed to providing our customers with highquality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about an Invitrogen product or service, contact our Technical Service Representatives. Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis. The company will replace, free of charge, any product that does not meet those specifications. This warranty limits Invitrogen Corporation s liability only to the cost of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. Invitrogen reserves the right to select the method(s) used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order. Invitrogen makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, please report it to our Technical Service Representatives. Invitrogen assumes no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose. 27

34 Purchaser Notification Introduction Use of the plenti6/tr Vector Kit is covered under the licenses detailed below. Limited Use Label License No. 5: Invitrogen Technology The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For products that are subject to multiple limited use label licenses, the terms of the most restrictive limited use label license shall control. Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Life Technologies is willing to accept return of the product with a full refund. For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008; Phone (760) or outlicensing@lifetech.com. continued on next page 28

35 Purchaser Notification, continued Limited Use Label License No. 51: Blasticidin/Blasticidin Selection Marker Limited Use Label License No. 108: Lentiviral Technology Blasticidin and the blasticidin resistance gene (bsd) are the subject of U.S. Patent No. 5,527,701 sold under patent license for research purposes only. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, California Phone (760) Fax (760) The Lentiviral Technology (based upon the lentikat system) is licensed from Cell Genesys, Inc., under U.S. Patent Nos. 5,834,256; 5,858,740; 5,994,136; 6,013,516; 6,051,427; 6,165,782 and 6,218,187 and corresponding patents and applications in other countries for internal research purposes only. Use of this technology for gene therapy applications or bioprocessing other than for non-human research use requires a license from Cell Genesys (Cell Genesys, Inc. 342 Lakeside Drive, Foster City, California 94404). The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or forprofit entity), including non-gene therapy research and target validation applications in laboratory animals. 29

36 Product Qualification plenti6/tr The structure of the plenti6/tr vector is verified by restriction enzyme digestion. Blasticidin Blasticidin is lot-qualified by performing a kill curve on Blasticidin-sensitive and resistant mammalian cell lines. Blasticidin-sensitive cells should be killed at all concentrations tested ( µg/ml) within 10 days after addition of Blasticidin. 30

37 References Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989). Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985). A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, Buchschacher, G. L., Jr., and Wong-Staal, F. (2000). Development of Lentiviral Vectors for Gene Therapy for Human Diseases. Blood 95, Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J.-K. (1993). Vesicular Stomatitis Virus G Glycoprotein Pseudotyped Retroviral Vectors: Concentration to a Very High Titer and Efficient Gene Transfer into Mammalian and Nonmammalian Cells. Proc. Natl. Acad. Sci. USA 90, Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Mol. Cell. Biol. 7, Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999). Lipofectamine TM 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells. Focus 21, Dull, T., Zufferey, R., Kelly, M., Mandel, R. J., Nguyen, M., Trono, D., and Naldini, L. (1998). A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 72, Emi, N., Friedmann, T., and Yee, J.-K. (1991). Pseudotype Formation of Murine Leukemia Virus with the G Protein of Vesicular Stomatitis Virus. J. Virol. 65, Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, Felgner, P. L. a., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337,

38 References Hillen, W., and Berens, C. (1994). Mechanisms Underlying Expression of Tn10 Encoded Tetracycline Resistance. Annu. Rev. Microbiol. 48, Hillen, W., Gatz, C., Altschmied, L., Schollmeier, K., and Meier, I. (1983). Control of Expression of the Tn10-encoded Tetracycline Resistance Genes: Equilibrium and Kinetic Investigations of the Regulatory Reactions. J. Mol. Biol. 169, Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., and Hanaoka, F. (1991). Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells. Exp. Cell Res. 197, Kimura, M., Takatsuki, A., and Yamaguchi, I. (1994). Blasticidin S Deaminase Gene from Aspergillus terreus (BSD): A New Drug Resistance Gene for Transfection of Mammalian Cells. Biochim. Biophys. ACTA 1219, Kjems, J., Brown, M., Chang, D. D., and Sharp, P. A. (1991). Structural Analysis of the Interaction Between the Human Immunodeficiency Virus Rev Protein and the Rev Response Element. Proc. Natl. Acad. Sci. USA 88, Landy, A. (1989). Dynamic, Structural, and Regulatory Aspects of Lambda Sitespecific Recombination. Ann. Rev. Biochem. 58, Lewis, P. F., and Emerman, M. (1994). Passage Through Mitosis is Required for Oncoretroviruses but not for the Human Immunodeficiency Virus. J. Virol. 68, Luciw, P. A. (1996) Human Immunodeficiency Viruses and Their Replication. In Fields Virology, S. E. Straus, ed. (Philadelphia, PA: Lippincott-Raven Publishers), pp Malim, M. H., Hauber, J., Le, S. Y., Maizel, J. V., and Cullen, B. R. (1989). The HIV-1 Rev Trans-activator Acts Through a Structured Target Sequence to Activate Nuclear Export of Unspliced Viral mrna. Nature 338, Naldini, L. (1999) Lentiviral Vectors. In The Development of Human Gene Therapy, T. Friedmann, ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press), pp